The first studies of recurrent amplicons in rhabdomyosarcoma (RMS) are focusing on the 12q13-q14 amplicon, which occurs preferentially in a subset of PAX3-FOXO1-positive RMS (25% of cases) and only in a smaller subset of PAX7-FOXO1-positive and fusion-negative cases. Our previous studies localized the minimal region of amplification to a 0.55 Mb region containing 28 genes, including the CDK4 proto-oncogene. Our subsequent expression profiling studies showed that 7 of these genes were consistently overexpressed at the RNA level in amplified RMS tumors. Collaborative studies with Dr. Javed Khan of the Pediatric Oncology branch applying high density array analysis and next generation sequencing has confirmed the size and distribution of this 12q13-q14 amplicon. To determine which of the 28 genes are overexpressed at the protein level, we set up a collaboration with Dr. Svetlana Pack and Dr. Stephen Hewitt of the Laboratory of Pathology. Two tissue microarrays of RMS specimens were obtained from the Children's Oncology Group (COG). Using RT-PCR and FISH assays, we determined the fusion status of the RMS specimens on these TMA's. Dr. Pack developed a set of fluorescence in situ hybridization probes to determine the amplification status of the 12q13-q14 region in cases on these TMA's; the findings from these FISH studies are consistent with our previous reported findings of the frequency of 12q13-q14 amplification in RMS fusion subsets. Dr. Hewitt is now working with my laboratory to analyze the expression status of the CDK4 protein in these TMA's by immunohistochemistry. To begin begin our analysis of the functional consequences of this amplification event, we first focused on the CDK4 gene. For these studies, we identified Rh30 as a fusion-positive RMS cell line with the 12q13-q14 amplicon. For comparison, we selected several fusion-positive RMS cell lines that lack the 12q13-q14 amplicon. In addition to analyzing these cell lines in culture, we developed a series of intramuscular xenografts from these cell lines as part of a collaboration with Dr. Chand Khanna of the Pediatric Oncology Branch. An antibody that detects CDK4 protein expression by western blot has been identified and successfully used. Western blot analysis with this antibody confirms increased expression of CDK4 in lines and corresponding xenografts with 12q13-q14 amplification. In one set of functional studies, a tetracycline-inducible lentiviral expression vector was provided by Dr. Ji Luo of the Medical Oncology Branch. We subcloned the CDK4 cDNA into this expression vector and transduced the expression construct into several RMS cell lines that do not have amplification of the 12q13-q14 region. Initial dose-response and time course studies in these transduced cells confirm that CDK4 protein expression is readily increased in transduced Rh28 cells treated with doxycycline in comparison to controls. The phenotype of these transduced cells is being assessed in cell culture studies of growth and transformation. In complementary studies, a set of lentivral shRNA expression constructs targeted against the CDK4 gene were transduced into Rh30 cells and demonstrated clear evidence of decreased CDK4 protein expression. Furthermore, the decrease in CDK4 expression in these cells is associated with substantial defects in cell growth and possibly survival. To further analyze this issue, IPTG-inducible shRNA constructs were developed and tested in Rh30. As predicted, these inducible constructs permit controlled inhibition of CDK4 expression in the Rh30 cells. We are now investigating the phenotypic consequences of varying levels of CDK4 expression in these cells. Among the fusion-positive RMS cells that do not have 12q13-q14 amplification, we selected Rh28 and Rh41 as representative lines with low and intermediate levels of CDK4 protein expression. These lines are being similarly tested with the IPTG-inducible shRNA constructs. Finally, an animal protocol has recently been approved by the Institutional Animal Care and Use Committee to extend our cell culture studies and assess the role of CDK4 in contributing to and maintaining tumorigenicity in laboratory animals.